Determination of Vitamin C (Ascorbic Acid) Using High … · 2016-09-14 · 1.3 Content of vitamin C in foods AA can be mostly found in fruits and vegetables. The main sources of

Sensors 2008, 8, 7097-7112; DOI: 10.3390/s8117097

sensors ISSN 1424-8220

http://www.mdpi.com/journal/sensors

Article

Determination of Vitamin C (Ascorbic Acid) Using High Performance Liquid Chromatography Coupled with Electrochemical Detection

Zbynek Gazdik 1,2, Ondrej Zitka 3, Jitka Petrlova 3, Vojtech Adam 3,4, Josef Zehnalek 3,

Ales Horna 5, Vojtech Reznicek 1, Miroslava Beklova 6 and Rene Kizek 3,*

1 Department of Breeding and Propagation of Horticultural Plants, Faculty of Horticulture, Valticka

337, CZ-691 44 Lednice, Faculty of Agronomy, Zemedelska 1, CZ-613 00 Brno, Mendel

University of Agriculture and Forestry, Czech Republic 2 Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, Faculty of

Agronomy, Zemedelska 1, CZ-613 00 Brno, Mendel University of Agriculture and Forestry, Czech

Republic 3 Department of Chemistry and Biochemistry, Faculty of Agronomy, Zemedelska 1, CZ-613 00 Brno,

Mendel University of Agriculture and Forestry, Czech Republic 4 Department of Animal Nutrition and Forage Production, Faculty of Agronomy, Zemedelska 1, CZ-

613 00 Brno, Mendel University of Agriculture and Forestry, Czech Republic 5 Tomas Bata University, T.G. Masaryka 275, CZ-762 72 Zlin, Czech Republic 6 Department of Veterinary Ecology and Environmental Protection, Faculty of Veterinary Hygiene

and Ecology, University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, CZ-612 42

Brno, Czech Republic

* Author to whom correspondence should be addressed; E-mail:[email protected]

Received: 4 August 2008; in revised form: 4 November 2008 / Accepted: 6 November 2008 /

Published: 7 November 2008

Abstract: Vitamin C (ascorbic acid, ascorbate, AA) is a water soluble organic compound

that participates in many biological processes. The main aim of this paper was to utilize

two electrochemical detectors (amperometric – Coulouchem III and coulometric –

CoulArray) coupled with flow injection analysis for the detection of ascorbic acid.

Primarily, we optimized the experimental conditions. The optimized conditions were as

follows: detector potential 100 mV, temperature 25 °C, mobile phase 0.09% TFA:ACN,

OPEN ACCESS

Sensors 2008, 8

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3:97 (v/v) and flow rate 0.13 mL·min-1. The tangents of the calibration curves were 0.3788

for the coulometric method and 0.0136 for the amperometric one. The tangent of the

calibration curve measured by the coulometric detector was almost 30 times higher than

the tangent measured by the amperometric detector. Consequently, we coupled a

CoulArray electrochemical detector with high performance liquid chromatography and

estimated the detection limit for AA as 90 nM (450 fmol per 5 µL injection). The method

was used for the determination of vitamin C in a pharmaceutical preparations (98 ± 2 mg

per tablet), in oranges (Citrus aurantium) (varied from 30 to 56 mg/100 g fresh weight), in

apples (Malus sp.) (varied from 11 to 19 mg/100 g fresh weight), and in human blood

serum (varied from 38 to 78 µM). The recoveries were also determined.

Keywords: Ascorbic Acid; Flow Injection Analysis; High Performance Liquid

Chromatography; Electrochemical Detection; Fruits; Pharmaceutical Preparation; Human

Blood Serum

1. Introduction

1.1 Biological function of vitamin C

Vitamin C (ascorbic acid, ascorbate, AA) is a water soluble organic compound involved in many

biological processes (Figure 1). AA plays crucial roles in electron transport, hydroxylation reactions

and oxidative catabolism of aromatic compounds in animal metabolism [1]. Although all the functions

of AA are not fully explained, it is likely that it is also involved in maintaining the reduced state of

metal cofactors, for example at monooxygenase (Cu+) and dioxygenase (Fe2+) [2]. In cells the other

role of AA is to reduce hydrogen peroxide (H2O2), which preserves cells against reactive oxygen

species [3-5]. An oxidation cycle of ascorbic acid to dehydroascorbic acid is shown in Figure 1. The

details about ascorbic acid antioxidant system cooperated with glutathione was described by Meister

[6]. Besides this, primates and several other mammals are not able to synthesise ascorbic acid [5]. The

animal species, which are able to produce this molecule, biosynthesise AA from glucose catalyzed L-

gulonolactonoxidase [1,2]. In spite of the ability to synthesize this molecule both groups of animal

species suffer from deficiency of AA [1,2].

1.2 Daily needs of vitamin C

The only way humans uptake ascorbic acid is via food [7], but the daily needs of vitamin C for a

human are not clear yet. Linus Pauling postulated that people's needs for vitamins and other nutrients

vary markedly and that to maintain good health, many people need amounts of nutrients much greater

than the recommended doses. According to his suggestions, daily uptake of vitamin C has to be within

units of grams of AA to reduce the incidence of colds and other diseases. These “huge” amounts of

AA have not been ever proved as the reason for large reducing of the incidence of illnesses. Nowadays

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the estimated average requirement and recommended dietary allowance of ascorbic acid are 100 mg

per day and 120 mg per day, respectively [8,9].

1.3 Content of vitamin C in foods

AA can be mostly found in fruits and vegetables. The main sources of AA are citrus fruits, hips,

strawberries, peppers, tomatoes, cabbage, spinach and others [3]. If one wants to uptake AA from

animal sources, liver and kidney are the tissues with highest contents of this molecule, but in

comparison with plant sources the amount of AA is very low [10]. The content of AA in food can be

affected by many factors such as clime, method of harvest, storing and processing. Thus, there is a

need of analytical procedures able to not only monitor AA content in agricultural and food products,

but also in body liquids and tissues [11]. Authors also paid their attention at detection of AA in blood

serum [12-15].

Figure 1. Scheme of a biological function of ascorbic acid (GSH – reduced glutathione,

GSSG – oxidized glutathione).

1.4 Methods for ascorbic acid determination

Many analytical techniques including sensors and biosensors [16-18] have been suggested for a

detection of ascorbic acid in very varied types of samples. Hyphenated instruments consisting of flow

injection analysis [19-22], high performance liquid chromatography [23-25] or capillary

electrophoresis [26-29] instruments and a detector are mostly utilized for the determination of AA.

However, some of these methods are time-consuming, some are costly, some need special training

H

C

C

C

C

C

C

H

H OH

OH

H

OH

OH

O

O

L-ascorbate(Vitamin C)

ascorbate oxidase

glutathione dehydrogenase

GSSG 2GSH

H2O½ O2

Cu 2+

2-dehydro-L-gulonolactone

non-

enzy

mat

ic

L-dehydroascorbate

ascorbate-2,3-dioxygenase

C

COOH

OOH

oxalate

L-threonate

Fe 2+

O2

2,3-dioxo-L-gulonate

H2O

H

C

C

C

C

C

C

H

H OH

OH

H

O

O

O

O

Sensors 2008, 8

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operators, or they suffer from the insufficient sensitivity or selectivity. Limits of detection ranged from

µM [30-32] to nM [33-36] and lower [12].

Electrochemical detection is an attractive alternative method for detection of electroactive species,

because of its inherent advantages of simplicity, ease of miniaturization, high sensitivity and relatively

low cost. Electrochemical detection typically worked in amperometric or coulometric mode can be

coupled with liquid chromatography to provide high sensitivity to electroactive species. The main aim

of this paper is to utilize two electrochemical detectors (amperometric – Coulouchem III and

coulometric – CoulArray) coupled with flow injection analysis for detection of ascorbic acid. The

more sensitive technique is further applied on analysis of real samples (pharmaceutical preparation,

oranges and apples fruits, and human blood serum).

2. Material and Methods

2.1 Chemicals, material and pH measurements

HPLC-grade acetonitrile (>99.9%; v/v) from Merck (Darmstadt, Germany) was used. Other

chemicals used were purchased from Sigma-Aldrich (St. Louis, USA) in ACS purity unless noted

otherwise. Stock standard solutions of the AA (100 mM) were prepared with ACS water (Sigma-

Aldrich, USA) and stored in the dark at -20 °C. Working standard solutions were prepared daily by

dilution of the stock solutions. The stability of AA in samples is strongly influenced by oxygen, which

oxidises AA to dehydroascorbic acid. To avoid direct an oxidation reducing agents or acidification by

acids can be used [37]. Here, we used dithiothreitol (DTT). All solutions were filtered through 0.45 μm

Nylon filter discs (Millipore, Billerica, Mass., USA) prior to HPLC analysis. The pH value was

measured using WTW inoLab (Weilheim, Germany), controlled by software MultiLab Pilot. The pH-

electrode was regularly calibrated with WTW buffers (Weilheim, Germany).

2.2 Flow injection analysis/High performance liquid chromatography with CoulArray or

Coulochem electrochemical detector

CoulArray. The FIA/HPLC-ED system consisted of two solvent delivery pumps operating in the

range 0.001-9.999 mL·min-1 (Model 582 ESA Inc., Chelmsford, MA), a reaction coil (1 m) and/or

Metachem Polaris C18A reversed-phase column (150.0 × 2.1 mm, 3 μm particle size; Varian Inc., CA,

USA) and a CoulArray electrochemical detector (Model 5600A, ESA, USA). The electrochemical

detector includes two flow cells (Model 6210, ESA, USA). Each cell consists of four analytical cells

containing working carbon porous electrode, two auxiliary and two reference electrodes. Working

electrodes were polished electrochemically applying of positive/negative potential cycles (1/-1 V) at

increased flow of the mobile phase (1 mL·min-1). Both the detector and the reaction coil/column were

thermostated. The sample (5 µL) was injected manually.

Coulochem III. The FIA-ED system consisted of a solvent delivery pump operating in range of

0.001-9.999 mL·min-1 (Model 583 ESA Inc., Chelmsford, MA, USA), a guard cell (Model 5020 ESA,

USA), a reaction coil (1 m) and an electrochemical detector. The electrochemical detector (ED)

includes one low volume flow-through analytical cells (Model 5040, ESA, USA), which is consisted

Sensors 2008, 8

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of glassy carbon working electrode, palladium electrode as reference electrode and auxiliary carbon

electrode, and Coulochem III as a control module. A glassy carbon electrode was polished

mechanically by 0.1 μm of alumina (ESA Inc., USA) and sonicated at the laboratory temperature for 5

min using a Sonorex Digital 10 P Sonicator (Bandelin, Berlin, Germany) at 40 W as it was described

by [38]. The sample (5 μL) was injected manually. The obtained data were treated by CSW 32

software. The experiments were carried out at room temperature.

2.3 Preparation of real samples

The pharmaceutical preparation (a tablet) – Celaskon (Leciva, Prague, Czech Republic) was

ground in a mortar (n = 5). Then, ground powder (about 1 mg) was dissolved in ACS water (1 mL).

Oranges and apples (Citrus aurantium and Malus sp.) were bought at TESCO stores (n = 5). The

pericarps of the fruits were removed, and then the fruits (app. 0.25 g) were homogenized using a

mortar. The extracts obtained were filtered through filter paper (Niederschlag, Germany), transferred

into a volumetric flask and diluted with ACS water. Measurements of the samples were carried out

immediately after preparation steps. Human blood serum samples were obtained from the Department

of Clinical Biochemistry, Trauma Hospital Brno (Czech Republic), (n = 10). Human sera were frozen

at –20 °C immediately after collection. The samples were 100 × diluted with ACS water and filtered

through 0.45 µm Teflon membrane filter prior to measurement.

2.4 Accuracy, precision and recovery

Accuracy, precision and recovery of AA were evaluated with homogenates (human blood serum, a

fruit and Celaskon tablets) spiked with the standard. Before extraction, AA standards (100 µL) and

water (100 µL) were added to the homogenates of real samples. The homogenates were assayed

blindly and AA concentrations were derived from the calibration curves. Accuracy was evaluated by

comparing estimated concentrations with known concentrations of AA. Calculation of accuracy (%

Bias), precision (% C.V.) and recovery was carried out as indicated by [39-41].

2.5 Descriptive statistics

Data were processed using MICROSOFT EXCEL® (USA). Results are expressed as mean ± S.D.

unless noted otherwise. The detection limits (3 signal/noise, S/N) were calculated according to Long

and Winefordner [42], whereas N was expressed as standard deviation of noise determined in the

signal domain unless stated otherwise.

3. Results and Discussion

Stationary and flow electrochemical techniques are very attractive instruments for determining

various biologically important compounds such as proteins [43-60], organic compounds of plant origin

[61-66], drugs [67-70], etc. Here, we aimed at utilizing two different electrochemical detectors –

Sensors 2008, 8

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amperometric (Coulouchem III) or coulometric (CoulArray) coupled with high performance liquid

chromatography for detection of ascorbic acid.

3.1 Flow injection analysis coupled with CoulArray electrochemical detector to detect ascorbic acid

An electrochemical behaviour of AA at the surface of working electrodes was investigated. FIA

enables us to optimise experimental conditions for analytical determination of AA easily and rapidly.

Primarily, the influence of potential applied to single working electrodes on oxidation signal of AA

was studied. The potential varied from 100 to 400 mV and signal of various concentration of AA

(12.5, 25, 50, 100, 200, 300, 400, 500 and 1000 µM) was measured.

Figure 2. FIA coupled with CoulArray electrochemical detector. FIA-ED full scan of

ascorbic acid (12.5, 25, 50, 100, 200, 300, 400, 500 and 1000 µM). Detector electrodes

potentials at full scan: 100, 150, 200, 250, 300, 350, 380 and 400 mV (A). Dependence of

ascorbic acid peak height on detector temperature (B), on content of acetonitrile in mobile

phase consisted from acetonitrile and trifluoroacetic acid (C) and on TFA concentration

(D). FIA-ED parameters: flow rate of mobile phase – 0.1 mL·min-1; ascorbic acid

concentrations – 100 µM; 5 µL samples was injected.

Each concentration belongs to the signal shown in FIA-ED record in Figure 2A. Apparently

maximal current responses were measured at the surface of the first working electrode. This

phenomenon was observed at all concentrations of AA measured by FIA-ED. Moreover it can be

concluded that lower concentration of AA was measured, the signal disappeared earlier (Figure 2A).

This phenomenon associates with i) type of detector used because CoulArray detector operates in

coulometry mode and coulometric detector promotes near 100 % electrochemical conversion of

analytes below the designated potential of the detector, and with ii) easy electrochemical oxidation of

AA at the surface of the working electrodes already under low potentials (about 100-200 mV). For

[400 mV]

[380 mV]

[350 mV]

[300 mV]

[250 mV]

[200 mV]

[150 mV]

[100 mV]

Res

pon

se (

50 A

)

FIA coupled with CoulArray electrochemical detector

A

6 (min)

[400 mV]

[380 mV]

[350 mV]

0

40

80

120

0 5 10Content of ACN ()

0

40

80

120

0.00 0.05 0.10 0.15

TFA concentration ()

Pea

k he

ight

(

)

C D

0

40

80

120

10 20 30 40

Detector temperature (°C)

B

Pea

k he

ight

(

)

Pea

k he

igh

t (

)

Sensors 2008, 8

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choosing of the optimal potential for detection of AA, we applied the potential scale from 100 to 400

mV per 50 mV at all eight electrodes and measured the signal at the first electrode only. Based on

these results, the most suitable potential for detection of AA was 100 mV.

Further, the affecting height of the signal of AA by temperature was studied. The temperature

within the range from 15 to 40 °C was tested. The dependence obtained is shown in Figure 2B. The

peak height increased for more than 30 % at 25 °C compared to signals obtained at lower temperatures

(15 and 20 °C). At temperatures higher than 25 °C very low changes in the height was observed

(Figure 2B). Therefore temperature of 25 °C was used in the following experiments.

It is commonly known that electrochemical analysis needs the presence of an electrolyte, although

the presence of a non-aqueous solvent in a mobile phase is needed for successful and rapid

simultaneous determination of compounds of interest. These non-aqueous solvent negatively influence

electrochemical analysis [41,71]. Therefore, affecting of AA signal by the ratio of

acetonitrile:trifluoroacetic acid (ACN:TFA, v/v) was investigated (Figure 2C). We did not determine

any changes in repeatability with increasing content of ACN in mobile phase. The highest signal was

measured at 3% (v/v) content of ACN in the mobile phase.

Figure 3. Effect of AA concentration. Dependence of peak height on different AA

concentrations (15, 30, 55, 85, 110 and 140 µM) (A). Human blood serum. Influence of

biological matrix on AA signal. Concentration of AA – 57, 114, 227 and 341 µM (B). FIA-

ED parameters were as follows: mobile phase – acetonitrile and 0.09% trifluoroacetic acid

in ratio 3:97; detector temperature – 25 °C; flow rate of mobile phase – 0.13 mL·min-1; 5

µL samples was injected.

It is a common knowledge that trifluoroacetic acid (TFA) is ion pairing agent in liquid

chromatography that can react with the vitamin and may assign them charge. Thus, we selected a

mixture of acetonitrile with aqueous solution of trifluoroacetic acid at ratio 3:97 (v/v) and tested the

influence of various concentrations of TFA. We found out that the response increased to 0.005%

concentration of TFA, then slightly decreased. The highest signal of AA was measured at 0.09%

TFA:ACN, 3:97 (v/v) (Figure 2D). Moreover, flow rate of the mobile phase is other important

parameter influencing peak height. We tested the flow of mobile phase from 0.05 to 0.2 mL·min-1. The

highest current responses were obtained at flow 0.13 mL·min-1.

y = 0.3788x - 0.1461

R2 = 0.99850

10

20

30

40

50

60

0 50 100 150

Pea

k he

ight

(A

)

Concentration of AA (mol.l-1)

y = 0.2897x + 0.6118

R2 = 0.9942

0

20

40

60

80

100

120

0 100 200 300 400

10 µA10 µA

1 min

15 mol.l-1

30 mol.l-1

55 mol.l-1

85 mol.l-1

110 mol.l-1

140 mol.l-1

1 min

15 mol.l-1

30 mol.l-1

55 mol.l-1

85 mol.l-1

110 mol.l-1

140 mol.l-1

Concentration of AA (mol.l-1)

Pea

k he

ight

(A

)

Effect of AA concentration

Human serum

A B

FIA coupled with CoulArray electrochemical detector

Sensors 2008, 8

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The optimal experimental conditions are: detector potential 100 mV, temperature 25 °C, mobile

phase 0.09% TFA:ACN, 3:97 (v/v) and its flow rate 0.13 mL·min-1. Under these conditions we

measured the dependence of AA peak height on its concentration. The peaks obtained were well

developed and symmetrical (insets in Figure 3A). The calibration curve is shown in Figure 3A (y =

0.3788x – 0.1461; R2 = 0.9985). The detection limit (3 S/N) for AA was evaluated as 100 fmol per 5

µL injection. Further the method was utilized for determination of AA spiked into human blood serum

samples. AA was spiked into blood serum (100 × diluted) and electrochemical responses were

observed. The obtained calibration curve (y = 0.2897x + 0.6118; R2 = 0.9942) is shown in Figure 3B.

The detection limit (3 S/N) was evaluated as 20 pmol for a 5 µL injection.

3.2 Flow injection analysis coupled with Coulochem III electrochemical detector to detect AA

Coulochem III is an amperometric detectors, which are somewhat inefficient because only a

fraction of analyte (typically 5–10 %), which passes over the working electrode, actually diffuses onto

the electrode surface and experiences electrochemical conversion [72]. One may suggest that

coulometric detector CoulArray should be 10–20 times more sensitive than amperometric detection

simply because of improved mass transfer. Unfortunately the increased conversion efficiency of the

analyte is accompanied by a similar increase for the electrolyte (background) reactions, and nearly no

lowering of detection limits is realized [72]. Moreover, amperometry can provide better LOD in terms

of concentrations with miniaturized cells. Therefore we adopted above optimized experimental

conditions to detect AA by FIA coupled Coulochem III electrochemical detector and aimed on

comparison of amperomeric and coulometric detectors. Unlike CoulArray amperometric detector

Coulochem contains a device called “Guard cell”, which can oxidize electroactive impurities in mobile

phase and, thus, lower the noise. We tested five potentials (-100, -50, 0, 50 and 100 mV) applied on

Guard cell and measured the signal of AA. The dependence obtained is shown in Figure 4A. Based on

the results obtained the optimal Guard cell potential was 0 mV. The potentials lower or higher than the

optimal value slightly decreased the signal of AA. This phenomenon can be associated to easy

electrochemical conversion of the target molecule already at Guard cell. To compare the detectors we

again measured the dependence of AA concentration on peak height (Figure 4B). The equation of the

calibration curve obtained was y = 0.0136x + 0.0189, with R2 = 0.9990.

The concentration range of AA analysed using both coulometric and amperometric detectors was

almost the same due to possibility of comparing of the sensitivity of the instruments. The tangents of

the calibration curves were 0.3788 for coulometric and 0.0136 for amperometric. Based on these

results the tangent of calibration curve for AA measured using coulometric detector was almost 30

times higher than the tangent measured by amperometric detector. Coulometric detector is much more

sensitive to presence of AA, thus, we utilized this detector in following experiments.

Sensors 2008, 8

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Figure 4. FIA coupled with Coulochem III electrochemical detector. Dependence of peak

height on Guard cell potentials (-100, -50, 0, 50 and 100 mV) (A) and different AA

concentrations (15, 30, 55, 85, 110 and 140 µmol·L-1) (B). FIA-ED parameters were the

same as shown in Figure 3.

Figure 5. HPLC-ED detection of ascorbic acid. HPLC-ED chromatogram of ascorbic

acid (10 µM) (A). Dependence of peak area on different AA concentrations in the range

from 0.5 – 20 mM (B) and 10 – 90 µM (C). HPLC-ED parameters were as follows:

chromatographic column – MetaChem Polaris C18A (150 × 2.0 mm, 3 µm particle size)

mobile phase – acetonitrile and 0.09% trifluoroacetic acid in ratio 3:97; detector

temperature – 25 °C; flow rate of mobile phase – 0.13 mL·min-1; all detectors potential

– 150 mV; 5 µL samples was injected.

3.3 High performance liquid chromatography coupled with CoulArray electrochemical detector to

detect AA

Under the optimized conditions mentioned above ascorbic acid was measured using high

performance liquid chromatography coupled with CoulArray electrochemical detector (HPLC-ED).

The retention time of AA was 5.4 min. (Figure 5A). Dependence of peak area on AA concentration

y = 0.0136x + 0.0189

R2 = 0.9990

0

0.3

0.6

0.9

1.2

0 20 40 60 80 100

1.29

1.30

1.31

1.32

-150 -100 -50 0 50 100 150

Concentration of AA (µM)

Pea

k h

eigh

t (µ

A)

FIA coupled with Coulochem III electrochemical detector

Guard cell potential (mV)

Pea

k h

eigh

t (µ

A)

A B

y = 24.921x + 12.043

R2

= 0.9967

0

200

400

600

0 5 10 15 20

Concentration of AA (µM)

Pea

kar

ea (

µC

)

Pea

kar

ea (

µC

)

4 5 6 7 8

Retention time (min)

4 5 6 7 8

Retention time (min)

5 μA

Ascorbic acid (10 M)

C

y = 0.0307x – 0.1417R2 = 0.9905

0

10

20

30

0 50 100

A B

HPLC-ED detection of ascorbic acid

Sensors 2008, 8

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was strictly linear and relative standard deviation (R.S.D.) was about 6 % (n = 5). Equation of

calibration curve measured within the range from 0.5 to 20 mM of AA was y = 24.921x + 12.043 with

R2 = 0.9967 (Figure 5B). In addition we attempted to analyse lower concentration range from 10 to 90

µM. The calibration curve obtained was y = 0.0307x – 0.1417; R2 = 0.9905 (Figure 5C). Using the

optimized HPLC-ED it was possible to detect nanomolar concentrations of AA (LOD: 90 nM; 450

fmol per 5 µL injection).

Figure 6. Typical HPLC-ED chromatograms of pharmaceutical preparation, extracts from

apples and oranges, and human blood serum. For HPLC-ED parameters see caption of

Figure 5.

3.4 Analysis of real samples

The concentration of ascorbic acid was determined in pharmaceutical preparation, two species of

fruits and human blood serum under the optimized experimental conditions using HPLC coupled with

CoulArray electrochemical detector. Typical HPLC-ED chromatograms of the fore-mentioned samples

are shown in Figure 6. We determined the concentration of ascorbic acid as 98 ± 2 mg per one tablet in

a pharmaceutical preparation called Celaskon. The manufacturer of this preparation declares the

amount of AA as 100 mg per tablet. The recovery of the amount of AA added into the sample was 105

% for lower addition of AA (5 µg·mL-1) and 95 % for higher addition of AA (15 µg·mL-1); for more

details see in Table 1. Moreover we used HPLC-ED to determine AA concentration in fruits species.

We found that AA amount in oranges (Citrus aurantium) varied in the range from 30 to 56 mg/100 g

of fresh weight and in apples (Malus sp.) from 11 to 19 mg/100 g of fresh weight. The recovery of AA

measured in the homogenate prepared from fruits Citrus aurantium was 103 % for lower addition (5

µg·mL-1) and 104 % for higher addition (15 µg·mL-1); Table 1. To evaluate HPLC-ED technique for

analysis of human body liquids we spiked human blood serum and found out that recovery of AA

0.5 μ A

Retention time (min)

5 µl injected

Serum without vitamin C

Hum

an b

lood

ser

um

4 6 84 6 8

1 μA

Serum with 5 µM vitamin C

Serum with 10 µM vitamin C

Serum with 15 µM vitamin C

Retention time (min)

5 μA

Analysis of real samples

Ora

nge

4 6 8

10 µl injected

5 µl injected

0.5 μ A

Retention time (min)4 6 8

5 µl injected

10 µl injectedA

pple

Pha

rmac

eutic

al p

repa

ratio

n

10 μA

Retention time (min)4 6 8

10 µl injected

5 µl injected

Retention time (min)

5 μA5 μA

Analysis of real samples

Ora

nge

4 6 84 6 8

10 µl injected

5 µl injected

0.5 μ A

Retention time (min)4 6 84 6 8

5 µl injected

10 µl injectedA

pple

Pha

rmac

eutic

al p

repa

ratio

n

10 μA10 μA

Retention time (min)4 6 84 6 8

10 µl injected

5 µl injected

Sensors 2008, 8

7107

varied from 102 to 98 % according to lower (5 µg·mL-1) or higher (15 µg·mL-1) content of AA. The

tested blood sera contained AA within the range from 38 to 78 µM.

Table 1. Recovery of AA for orange fruit (Citrus aurantium), Celaskon tablet and human

serum sample analysis (n = 3).

Sample Homogenate

(µg mL-1)a, b, c

Spiking AA

(µg mL-1)a, b

Homogenate + spiking

AA (µg mL-1)a, b

Recovery

(%)

Celaskon (tablets) 9.8 ±� 0.2 (2.0) 5.0 ± 0.2 (4.0) 15.6 ± 0.9 (5.8) 105

15.9 ± 0.9 (5.7) 24.3 ± 1.9 (7.8) 95

Citrus aurantium 4.3 ±� 0.1 (2.3) 5.1 ± 0.1 (1.9) 9.7 ± 0.3 (3.1) 103

15.3 ± 1.0 (6.5) 20.3 ± 1.6 (7.9) 104

Human serum 7.1 ±� 0.3 (4.2) 4.8 ± 0.2 (4.2) 12.1 ± 0.6 (5.0) 102

15.1 ± 0.9 (6.0) 21.7 ± 1.8 (8.3) 98 a amount of AA b the results are expressed as mean ± S.D. (C.V. %) c re-computation of AA molar concentration on weight concentration – 57 µM is 10 µg·mL-1

4. Conclusions

High performance liquid chromatography coupled with an eight channel electrochemical detector

appears to be a very suitable analytical instrument for sensitive ascorbic acid determination. Using the

optimized technique ascorbic acid was determined in pharmaceutical preparations, fruits and human

blood serum samples.

Acknowledgements

Financial support from MSMT 6215712402 and NAZVA QH8223 is greatly acknowledged.

References

1. Velisek, J.; Cejpek, K. Biosynthesis of food constituents: Vitamins. 2. Water-soluble vitamins:

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